Construction And Biological Characteristics Of Recombinant Fowl Adenovirus Serotype 4

Hepatitis-hydropericardium syndrome is caused by fowl adenovirus serotype 4(FAdV-4),which is mainly characterized by an accumulation of straw-colored fluid in the pericardial sac,and discolored and hepatitis.Since 2015,the highly pathogenic FAdV-4,which is prevalent in China,has caused serious economic losses to the poultry industry.However,the virulence genes of fowl adenovirus serotype 4 are still unclear.To investigate the effect of ORF17 gene on FAdV-4 replication and virulence,the recombinant virus with ORF17 gene deletion was constructed by homologous recombination technique and FAdV-4 strain HN/151025 strain was used as parental virus.First,the transfer vector pΔORF17-EGFP was transfected into LMH cells infected with HN/151025 to construct recombinant virus rHN-ΔORF17-EGFP.Then the EGFP expression cassette was removed,and rHN-ΔORF17 was obtained by reverse plaque screening.The rHN-ΔORF17 was serially passaged on LMH cells for 20 passages,and PCR was performed every 5passages.The results showed that rHN-ΔORF17 was genetically stable.The growth curve of the two recombinant viruses was consistent with the HN/151025 and the virus titer reached the peak at 96 h after inoculation.The virus titer of rHN-ΔORF17(2.22×10~8 PFU/mL)was similar to the parental virus HN/151025(4.26×10~8 PFU/mL),indicating that the deletion of ORF17 gene did not affect the virus replication ability;while the rHN-ΔORF17-EGFP titer(1.53×10~6 PFU/mL)decreased significantly and it was significantly different from the parental virus HN/151025(P<0.05).Comparing with the Canadian ON1 attenuated strain,the Chinese isolates FAdV-4 lack the 1966-bp(ORF19 and ORF27)in the 3′-end of genome.To understand the effect of this natural deletion on the virulence of FAdV-4,recombinant virus rHN-Δ35430-EGFP expressing EGFP was constructed by using the natural deletion strain at the position of 35430-35431 in 3′-end of the genome HN/151025 as parental virus.Then 1966-bp sequence,derived from the ON1 strain,was replaced with the EGFP expression cassette to construct a recombinant virus rHN-Δ35430-ON1.rHN-Δ35430-ON1 was serially passaged on LMH cells for 20 passages and PCR was performed every 5 passages.The results showed that rHN-Δ35430-ON1 was genetically stable.The growth kinetics showed that the replication kinetics of the two recombinant viruses were consistent with the HN/151025 and the virus titer reached the peak at 96 h after inoculation.The titer of rHN-Δ35430-EGFP was 6.8×10~7PFU/mL,and the titer of rHN-Δ35430-ON1(2.66×10~8 PFU/mL)was similar to that of the parental virus HN/151025(4.26×10~8 PFU/mL),indicating the 1966-bp sequence does not affect the viral replication.To evaluate the pathogenicity of rHN-ΔORF17 and rHN-Δ35430-ON1,fourty 28-day-old SPF chickens were randomly divided into 4 groups.Three groups were challenged with rHN-ΔORF17,rHN-Δ35430-ON1 and HN/151025 by intranasal and ocular routes.The challenged dose was 0.1 mL 8×10~5PFU.The control group was an equal volume of sterile PBS,and the symptoms of the chickens were observed continuously for 15 days after challenge.3 days after challenge,the chickens in the rHN-ΔORF17 and HN/151025 groups began to develop clinical symptoms and the chickens in the rHN-Δ35430-ON1 group developed clinical symptoms 4 days after challenge.The challenged chickens showed different signs,including depression,lethargy,neck narrowing,huddling with ruffled feathers.The incidence rate of the challenged groups was 100%and the peak incidence was 4~6 d after challenge.14days after challenge,the chickens gradually began to recover.The mortality was 10%in the HN/151025group,20%in the rHN-ΔORF17 group,10%in the rHN-Δ35430-ON1 group and the control was no death.Pathological necropsy was performed on the dead chickens and the three challenged groups all showed pericardial effusion,yellow necrosis of the liver and kidney enlargement.There were no significant differences between the three groups.Antigen was detected in the liver of the three challenged groups by immunohistochemistry.After challenged,throat swabs and serum were collected every 4 days from 4 d to 28 d to detect detoxification and antibody levels in chickens.100%chickens were detoxified from 4 d to 20 d.,The detoxification decreased at 24 d,and the three groups showed the same performance.In the serum antibody test,there was no difference in the overall antibody level and only the antibody positivity rate was different.The serumconversion rate of HN/151025 group was up to 88.9%,rHN-ΔORF17 group was 50%,and the rHN-Δ35430-ON1 was 33.3%.After the second challenge on the 32 d,there were no obvious clinical symptoms in the three groups.There was no significant difference in antibody positivity.he pharyngeal anal swab detoxification,the detoxification rate was 100%at 4~27 d,and increased after34~55 d,and the detoxification rate increased to 100%at 62 d.The three groups showed the same performance and no significant difference.Three groups all showed that detoxification repeated and the detoxification period was long.The results indicated that there was no difference in pathogenicity among the three groups.In conclusion,the ORF17 gene deletion and the 1966-bp sequence insertion have no effect on viral replication and pathogenicity.